(Adapted from the applicant's abstract) Activity of CFTR CL channels determines the composition of fluid that lines the airway surface. In cystic fibrosis (CF), the loss of this regulated channel disrupts normal electrolyte transport and may contribute directly to disease pathogenesis. Earlier work showed that the activity of CFTR CL channels in airway epithelia is regulated by changes in cellular levels of cAMP. An increase in cAMP leads to phosphorylation of CFTR's regulatory (R) domain which allows the channel to open and mediate transepithelial CL movement. This project focuses on the mechanisms by which the cytosolic R domain controls the CFTR channel. Preliminary data showed that addition of an R domain peptide (R1, containing 190 residues) to a CFTR variant in which much of the R domain had been deleted (CFTR-R/S660A) stimulated activity. However, un-phosphorylated R1 had not effect. These results show a novel mechanism of channel upregulation by a phosphorylated peptide. They also provide us with a unique system for studying the structure and function of the R domain and CFTR. The project will answer three question. 1) What parts of the R domain control the activity of CFTR? The investigators will test the hypothesis that smaller peptides from the R domain stimulate activity of CFTR-R/S660A. Conversely, they will delete smaller portions of the R domain from CFTR to learn how channel gating is altered. These two complimentary approaches will allow them to identify key functional and structural features of the domain to discover their effect on activity. 2) What is the structure of the R domain? We currently lack molecular level data about the three-dimensional structure of CFTR or any of its domains. Their preliminary data show that R domain peptide is soluble and functionally active. Thus, now is the time to solve the R domain structure. Guided by their functional studies, they will determine the high-resolution structure of functionally active R domain peptides using NMR spectroscopy. They will also test the exciting hypothesis that phosphorylation changes the structure. 3) How do alterations in R domain structure affect function? The information obtained in the first two specific aims will provide valuable framework of understanding and a model of the R domain. They will test that model using site-directed mutagenesis to address very specific questions about how structure determines function. The prospects for success in this project are enhanced by the combined electrophysiologic, biochemical, and structural skills of this unique team of investigators.